Cells in most tissues are interconnected by cell-cell channels which allow the passage of electrolytes and small molecules from cell to cell. Aggregates of these channels are contained in the clustered intramembranous particles of gap junctions. In excitable cells these channels are instrumental for impulse propagation, in other tissues they are thought to pass signal molecules and to synchronize the cells metabolically. The objective of this research project is to understand the process of formation of cell-cell channels. This problem is addressed by functional expression of cell-cell channels in paired oocytes and of gap junction hemichannels in single oocytes from cloned gap junction (connexin) cDNA. The oocyte assay will be used in combination with mutagenesis, site-specific reagents, tracer flux measurements, and the patch clamp technique to obtain information about molecular domains involved in various aspects of the channel formation process. Particular emphasis is given to the identification of pore lining amino acids. Domain exchange between two, distinct, open hemichannel forming connexins will be used to identify determinants of channel conductance and permeability. Observations on the voltage gating of single cx46 hemichannels has led to the hypothesis that the voltage gate's function could be the regulation of passage of intermediate sized molecules like second messengers while preserving electrical continuity between cells. This hypothesis will be tested by assessing the effect of voltage on the flux of second messengers and of fluorescent tracer molecules through the channels. In addition the pore size and its possible modulation by voltage will be tested by analyzing the accessibility of non-electrolytes to the channel.